Over the past 3 decades the rapid progression of botulinum exotoxin, the most toxic protein known, from experimental treatment of functional disorders to one of the most common aesthetic procedures performed today is remarkable. From the clinical research trials for the treatment of strabismus in the 1970s by Alan Scott to preoperative injections to improve wound healing aesthetics, botulinum toxin (BoTX) continues to find additional uses¹. Following the clinical observations that blepharospasm patients treated with BoTX had improvement of periocular rhytids, Carruthers in 1987 published the first paper describing the treatment of frown lines using BoTX. As clinical experience with these neurotoxins has expanded, the popularity and range of treatment indications have grown. Since the Carruthers landmark article, hundreds of articles have been published describing functional and aesthetic applications of BoTX. Botulinum toxin has many applications for improving facial appearance. These include kinetic facial wrinkle lines, protrusion of muscle bulk, and alteration of facial units controlled by the balance of elevator and depressor muscles. Aesthetic injection of botulinum toxin type A is approved by the U.S. Food and Drug Administration (FDA) for the glabellar region, while botulinum toxin type B is not FDA approved for aesthetic use. All injections described in this chapter outside the glabella are “off-label” and should be discussed with patients prior to injection.

There are several types of facial wrinkling that become more pronounced with age; gravitational, volume loss, redundancy, loss of skin elasticity, sleep creases, and dynamic facial lines—but only dynamic lines induced by muscle contraction are amenable to BoTX treatment. These kinetic facial wrinkles occur perpendicularly to the force of muscle contraction. It is theorized that repeated creasing of the skin in a predictable pattern induces changes in the dermal structure resulting in the eventual formation of a crease line at rest as well as deepening of the crease when the muscle is contracted. Botulinum toxin causes an amelioration of these dynamic lines by weakening the force of muscle contracture. When injected, the toxin decreases the release of acetylcholine at the presynaptic terminal of the neuromuscular junction and thereby decreases the strength of muscle contraction.

Neurotoxins produced by the gram-positive, anaerobic, sporulating Clostridium botulinum are the most potent toxins known to man and are the causative agents of botulism. Seven distinct antigenic botulinum toxins (BoTX-A, B, C, D, E, F, and G) produced by different strains of Clostridium botulinum have been described. The human nervous system is susceptible to serotypes A, B, E, F, and G, and unaffected by serotypes C and D.^2,3,4 All serotypes act preferentially on the peripheral nervous system where they inhibit release of acetylcholine from the presynaptic terminal of the neuromuscular junction. At higher doses, toxins may bind to nerve terminals at autonomic cholinergic ganglia with autonomic effects.⁵ Botulinum toxin may also have inhibitory effects on the release of other neurotransmitters such as substance p.

Botulinum toxin’s mechanism of action includes a three step process: binding, internalization, and neuromuscular blockade. It is not well understood how long the three-step process takes. With BoTX-A clinical expression of the effects typically takes 24 to 48 hours, and maximal muscle weakening is seen by 1 week (Botulinum toxin type B offers more rapid onset of action, but at a cost of shorter duration of action and greater diffusion at the site of injection). The first step is the irreversible binding of BoTX-A to presynaptic cholinergic receptors via the H chain’s 50 kDa carhoxy-terminal.^6,7,8,9 The second step involves internalization of the neurotoxin through a receptor-mediated endocytosis.¹⁰ The third step is neuromuscular blockade within the synapse.^11,12

Protein isoforms form a complex platform for docking and fusion of acetylcholine vesicles to the cell membrane that is necessary prior to release. These protein isoforms include vesicle associated membrane protein (VAMP, also known as synaptobrevin), synaptosomal associated protein (SNAP-25), and syntaxin. Alterations of these membrane proteins prevent the binding of the vesicles that release acetylcholine. Specific botulinum toxin serotypes act at different locations on these docking proteins. BoTX-A acts at SNAP-25, whereas BoTX-B acts at VAMP. The more profoundly the membrane proteins are structurally altered, the sooner they are replaced, and the faster the effects of the toxin abate. This may explain some of the difference in duration of effect of the different serotypes. While still inactivating the docking process, the longer acting serotypes may not alter the molecule significantly enough that the cell immediately recognizes that it must be replaced.

A great degree of injection variability exists, even among experienced physicians. Reconstitution techniques, concentration, total dose, location of injection, and depth of injection are examples of variables that may he altered. The size of the patient’s muscles, degree of facial dynamic action, previous response to injection, and desired extent of postinjection paresis are factored into individual dosing and injection patterns. Despite all these variabilities, certain tenants remain constant. Until the synapses are saturated, increasing the dose increases the degree of paresis. Increasing either the total dose or the volume at a given location will increase diffusion. The molecule has its effect at the neuromuscular junction, so it should be injected at or near this site.

Dilution techniques and injection materials also vary. Botox® is supplied in bottles of 100 U, and the amount of saline added to the bottle determines the concentration of the solution. The product insert recommends unpreserved saline for dilution, but recent studies have suggested that preserved saline may cause less discomfort with injection and that the preservative chemicals do not disrupt the action of the botulinum toxin molecule.²¹ Some treatment techniques use the same concentration of solution at all locations, but other techniques will vary the concentration at different locations. This may be accomplished by altering the dilution in the bottle or by changing the concentration in the syringe. Altering the concentration in the syringe may result in uneven dilution, but this has not found to be clinically troublesome. Injections may be given with a one cc syringe with a 30-gauge needle, or with a one-piece 1 cc insulin syringe. Insulin syringes have the advantage of less wasted material in the needle hub, but have the disadvantage that the needle cannot he changed if it becomes dull or contaminated. Although the manufacture recommend using the reconstituted BoTX-A within 4 hours after reconstitution, some authors have demonstrated equivalent efficacy after refrigeration for weeks and even freezing for up to six months.^{22,23,24,25,26}

Botulinum toxin type B (Myobloc^TM) is packaged in solution at a concentration in 500 U/0.1 cc. The purified liquid formulation is in a ready-to-use solution at a pH of 5.6. It does not need to be refrigerated and is stable at room temperature for up to 9 months (if refrigerated it is stable for up to 36 months). It is FDA approved for the treatment of cervical dystonia (spastic torticollis).²⁷ It may also be used to treat facial dystonias in patients who are nonresponsive to BoTX-A. The higher acidity increases discomfort on injection. This solution may be buffered, but this may dilute the concentration. With its greater diffusion, many physicians limit aesthetic use of BoTX-B to safer areas, such as the forehead region.

Skin preparation varies from no prep to 30- to 45-minute pretreatment with topical anesthetic and sterile cleansing techniques. For aesthetic patients the discomfort of the injections often does not warrant the time necessary for the topical aesthetic to work, but for some it makes the injection less unpleasant. Alcohol may denature the botulinum toxin, so for that reason, some physicians prefer to use betadine to clean the skin. A marking pen may be used to identify the locations of the injections. This helps the physician track the locations while injecting and assists the scribe in noting the locations in the chart. Many cosmetic patients are discriminating observers and will appreciate the opportunity to influence the design of their treatment pattern. Use a brand of skin-marking pen that washes off easily. Dosing and concentration information in the treatment techniques are based on Botox®. This information can be altered to apply Dysport or Myobloc once appropriate dosing ratios have been determined. Transient adverse effects such as injection site pain, bruising, headache, nausea and flulike symptoms may be experienced with BoTX injection. Absolute contraindications to BoTX injection include previous allergic responses to BoTX injections (or to human albumin) and injection into areas of infection. BoTX safety has not been established in pediatric patients (though its use in treating pediatric patients is well described) or pregnancy (category C). Facial asymmetry, and lid and brow malpositions are temporary and generally site and dose specific and should be discussed relative to the injection plan. Relative contraindications may include peripheral neuropathic diseases and neuromuscular diseases as well as certain medications (calcium channel blockers, and aminoglycosides) where the effects of BoTX may be less predictable.²³